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R G Vile

Publications and source records attributed to R G Vile.

At least 19 recordsLinked to original sources
PubMed1997

Generation of an anti-tumour immune response in a non-immunogenic tumour: HSVtk killing in vivo stimulates a mononuclear cell infiltrate and a Th1-like profile of intratumoural cytokine expression.

Direct delivery of the herpes simplex virus thymidine kinase (HSVtk) gene, in combination with the prodrug ganciclovir (GC), has been used for the treatment of localised, inoperable tumours. Several groups have shown that when rodent tumours are ablated in vivo with suicide genes, anti-tumour immunity can also be generated. Hence, this approach may also be useful in treating disseminated disease. Here we have studied the mechanisms associated with this anti-tumour immunity. In B16 HSVtk+ tumours being killed in vivo with GC treatment, we observed the induction of a pronounced intratumoural infiltrate of macrophages, CD4+ and CD8+ T cells. In addition, using reverse transcriptase polymerase chain reaction, expression of interleukin (IL)-2, IL-12, interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and granulocyte/macrophage colony-stimulating factor (GM-CSF) but not IL-4, IL-6 or IL-10, was observed, a profile of cytokine expression which resembles that of a Th1 immune response. To complement these findings, we also investigated the mechanisms by which expression of HSVtk leads to cell death. Our data show that B16/HSVtk+ cells die predominantly by necrosis, rather than apoptosis, on exposure to GC, a process which may be associated with the generation of anti-tumour inflammatory responses. From these data we propose a model for the induction of anti-tumour immunity using suicide genes and discuss the development of improved vectors for gene therapy to augment these effects in vivo.

Animals↗
PubMed1996

Expression of co-stimulatory molecules by tumor cells decreases tumorigenicity but may also reduce systemic antitumor immunity.

Many tumor cells do not express co-stimulatory molecules, and this may account, in part, for their poor ability to stimulate T cells directly. One strategy to enhance immune recognition would be to express such molecules on the tumor cell. Here, we show that expression of a member of the B7 family of co-stimulatory molecules by CMT93 murine colorectal tumor or 1735 murine melanoma cells resulted in a local antitumor response in immunocompetent mice. The antitumor effect was diminished in athymic nude mice, indicating that T cells played an important part in this response. The ability of the B7-expressing tumor cells to generate systemic protective immunity was investigated by excision of tumors that developed from the initial inoculation followed by rechallenge with parental tumor cells. CMT93 is a poorly immunogenic tumor and no significant systemic immunity was elicited by the expression of B7-1 in these cells. 1735 melanoma is a mildly immunogenic tumor. Unexpectedly, the systemic immunity obtained with 1735 tumors expressing B7-1 or B7-2 was weaker than that generated by parental 1735 cells (p < 0.001, stratified logrank test), even when coexpression of interferon-gamma in the B7-1 cells produced high levels of surface MHC class I expression. These results suggest that some caution is appropriate when considering the use of these molecules in the gene therapy of cancer.

Animals↗
PubMed1996

Immunotherapy III: Combinatorial molecular immunotherapy--a synthesis and suggestions.

Animal models have clearly shown that tumor cells may be amenable to molecular manipulation which can result in immune activation and rejection of unmodified cells (Chapters 4 and 5). The challenge now is to design clinical trials which have a realistic chance of success, (although the definition of 'success' is itself an important issue [see Chapter 9]. How should such a strategy be formulated? A review of the previous fifteen years since the first (immune) gene transfer studies were reported, encompasses a great wealth of data. Unfortunately, far from crystallising a set of unifying principles, these diverse reports shroud us in a fog of uncertainty as to how best to proceed. However, if this technology is to have practical, widespread application in the treatment of cancer patients, it is necessary to identify certain critical immunological goals which any protocols should achieve. Clear elucidation of these goals, by unifying the huge amount of disparate experimental data, must eventually be accomplished. In this chapter, we have reviewed the literature covering the era of molecular immunotherapy. We propose four general goals around which widely applicable clinical protocols, not necessarily dependent upon tumour type or experimental bias, might be based and suggest how they may be achieved in the context of gene transfer.

Animals↗
PubMed1996

Gene therapy for cancer, the course ahead.

Previous chapters have shown that there are a large number of therapeutic approaches under consideration for the gene therapy of cancer. Many of these have progressed into Phase I clinical trials. However, many of the early results are perceived to be disappointing in terms of low levels of gene transfer and systemic efficacy. In this concluding chapter, possible solutions to this state of unease are addressed, so that gene therapy of cancer can resume on its course to become a major contributor to clinical oncology in the years ahead.

Forecasting↗
PubMed1996

Selectin ligands on human melanoma cells.

Twelve established human melanoma lines were screened for surface expression of the carbohydrate antigens Lewisa (Lea), sialyl Lewisa (SLea), dimeric sialyl Lewisa (diSLea), sialyl LewisX (SLex) and dimeric sialyl LewisX (diSLeX). None of the lines expressed SLex, but 11/12 were positive for diSLeX and 7/12 were positive for SLea. Although both diSLeX and SLea have been reported to bind to E-selectin, none of the melanoma lines exhibited E-selectin-dependent adhesion to activated human umbilical vein endothelial cells (HUVECs). Three melanoma lines infected with a retroviral vector carrying the cDNA for the human Lewis fucosyltransferase (FucT-III) subsequently expressed SLeX at their cell surface and exhibited E-selectin-dependent adhesion to activated HUVECs. Treatment of these transduced cells with inhibitors of O-linked or N-linked protein glycosylation significantly inhibited E-selectin-mediated adhesion, though fluorescence-activated cell sorter analysis indicated no decrease in cell surface expression of SLeX, Slea or diSLeX. This suggests that the majority of SLeX/SLea-type glycans endogenously produced by human melanoma cells are not protein-associated and do not mediate E-selectin-dependent adhesion. These results support the hypothesis that E-selectin-dependent adhesion requires presentation of SLeX-type moieties on appropriate glycoproteins.

Carbohydrate Sequence↗
PubMed1996

Retroviral vectors. From laboratory tools to molecular medicine.

The majority of clinical trials for gene therapy currently employ retroviral-mediated gene delivery. This is because the life cycle of the retrovirus is well understood and can be effectively manipulated to generate vectors that can be efficiently and safely packaged. Here, we review the molecular technology behind the generation of recombinant retroviral vectors. We also highlight the problems associated with the use of these viruses as gene therapy vehicles and discuss future developments that will be necessary to maintain retroviral vectors at the forefront of gene transfer technology.

Animals↗
PubMed1996

Replication-competent retrovirus produced by a 'split-function' third generation amphotropic packaging cell line.

We report the discovery, on routine screening, of a replication-competent retrovirus (RCR) produced from a third generation amphotropic packaging cell line, GP + envAM12. In this line, the gag-pol and env helper genes are located on separate plasmids to minimise the chances of recombination events that may lead to RCR formation. Plasmid pBabeNeo was transfected into GP + envAM12 to obtain a producer line. Supernatant from this line was used to infect two lines, B16 and 1735-puro, and stable colonies which were obtained were pooled. Supernatants from both these lines were able to transfer the neo gene to fresh cells through several passages, indicating the presence of helper virus. Rescue of puro from 1735-puro cells, which contain the pBabePuro provirus, was also achieved. Mobilisation of lacZ from TELCeB6 cells, which contain the MFGnlslacZ provirus, was also possible. The RCR was able to transfer lacZ to ampli-GPE cells, an ecotropic packaging cell line, but transfer to fresh GP + envAM12 cells was minimal, demonstrating receptor interference of the RCR by the amphotropic envelope. These observations strongly suggest that a RCR with an amphotropic envelope arose in the GP + envAM12-pBabeNeo producer line. To our knowledge, this is the first report of RCR arising from routine use of GP + envAM12 cells or any similar third generation packaging line and has important implications for the use of such lines in human gene therapy protocols.

3T3 Cells↗
PubMed1995

Tissue-specific gene expression from Mo-MLV retroviral vectors with hybrid LTRs containing the murine tyrosinase enhancer/promoter.

Transcriptional tissue specificity was engineered directly into Moloney Murine Leukaemia Virus (Mo-MLV)-derived retroviral vectors by replacing the viral enhancer in the 3' long terminal repeat (LTR) with two different lengths (2.5 kbp or 769 bp) of the murine tyrosinase promoter/enhancer. The hybrid tyrosinase-LTR was transferred to the proviral 5' LTR following viral packaging and infection of target cell lines. Hybrid tyrosinase-LTR-driven IL-2 production was barely above background levels in infected nonmelanoma cell lines containing intact provirus, whereas infected melanoma cell lines expressed high levels of IL-2 and the larger tyrosinase promoter/enhancer fragment directed higher levels of transgene expression. By replacing the viral enhancer with the tyrosine promoter/enhancer sequences, promoter interference effects which we have previously observed when the tyrosinase promoter was included as an internal promoter within a similar retroviral vector were effectively abolished. Our data show that the hybrid tyrosinase-LTR behaves as a tightly regulated melanocytic-specific regulatory element when embedded in an enhancer-deleted Mo-MLV LTR. The use of other heterologous cellular promoter/enhancer elements in similar vectors should allow the development of simpler, targeted retroviral vectors for the expression of genes in selected cell types and may eventually provide for the development of safer, more efficient vectors for use in human gene therapy.

3T3 Cells↗
PubMed1995

Retroviruses as vectors.

Recombinant retroviruses have long been used to deliver heterologous genes to mammalian cells. Convenient packaging cell lines and vector plasmids have been distributed widely and 'home-made' retroviral vectors have now become a useful research tool in many laboratories. Compared to more traditional methods of gene transfer, retroviral vectors are extraordinarily efficient gene delivery vehicles which cause no detectable harm as they enter their target cells. In the nucleus the retroviral necleic acid becomes integrated into chromosomal DNA, ensuring its long-term persistence and stable transmission to all future progeny of the transduced cell. Up to 8 kilobases of foreign gene sequence can be packaged in a retroviral vector and this is more than enough for most gene therapy applications. Retroviral vectors can also be manufactured in large quantities to meet very stringent safety specifications. They have therefore been selected as the vectors of choice in 80% of the clinical gene therapy trials that have been approved to date. So far there have been no reported short- or long-term toxicity problems associated with their use in human gene therapy trials, now dating back to 1989. However, despite this impressive record, there is still great scope (and need) for the development of new, improved retroviral vectors and packaging systems to fuel further advances in the field of human gene therapy. In the following discussion, existing retroviral vectors are reviewed and current areas of technological development are emphasised.

Gene Expression Regulation, Viral↗
PubMed1995

Targeted gene therapy.

Melanin biosynthesis is limited to melanocytes partly as a consequence of transcriptional regulation of the enzymes involved in this pathway. Promoter sequences of these enzyme genes may be utilised to drive expression of complementary DNA coding for therapeutic genes so as to provide transcriptional targeting. We have used the 5'-flanking sequences of the murine tyrosinase or tyrosinase-related protein 1 (TRP-1) genes to show that such transcriptional targeting can be achieved both in vitro and in vivo. Using IL-2 as an example of an immunostimulatory gene and Herpes Simplex Virus thymidine kinase (HSVtk) as an example of a prodrug-activating gene we have shown, in murine model systems, that substantial anti-tumour effects can be achieved by targeted gene therapy approaches. The stage now is set for initial clinical evaluations in human patients.

Gene Targeting↗
PubMed1995

Effects of modulators of tyrosinase activity on expression of murine interleukin-2 cDNA driven by the tyrosinase promoter.

Sequence analysis of the promoter region of the murine tyrosinase gene identified various consensus motifs including AP2 sites, cAMP and TPA response elements (CREs/TREs) and retinoic acid response element (RARE) half-sites. By linking two different promoter lengths (2.5 kb or 769 bp) to murine interleukin-2 (IL-2) cDNA we have used IL-2 production by transduced B16 cells to monitor response to inducing agents capable of acting through these elements. Aminophylline or theophylline (0.1-2 mM) added to the culture medium of transfected B16, but not 3T3, cells, increased IL-2 secretion significantly (P > 0.05) in a dose-dependent fashion. This response was comparable in cells transfected with either the full length or the truncated promoter. Therefore, the cAMP responsiveness of the tyrosinase promoter probably is mediated by CREs and not AP2 sites, since the truncated promoter contains the former but not the latter regions. Retinoic acid at various concentrations (0.1-1 microM) evoked a standard increase in IL-2 production. Responses were similar for both promoter constructs, which suggests either that each RARE half-site can confer the full retinoic acid response, or that retinoic acid is mediating its effect through pathways independent of the RARE sites. TPA (2 nM-2 microM) had no effect on IL-2 production. These results demonstrate that the tyrosinase promoter can be induced by certain pharmacological agents and raise the possibility that administration of such substances may enhance expression of therapeutic genes controlled by this promoter.

3T3 Cells↗
PubMed1994

Systemic gene therapy of murine melanoma using tissue specific expression of the HSVtk gene involves an immune component.

Previously we have demonstrated safe and effective transfer of the HSVtk cytotoxic gene to primary murine melanoma tumors by direct injection of plasmid and retroviral vectors in which the HSVtk gene is driven by the tissue-specific tyrosinase promoter. However, for general clinical application such forms of therapy should, ideally, be effective against disseminated metastases. We report here that the number of recently established lung metastases of B16 melanoma in C57BL mice treated with ganciclovir is reduced compared to controls after multiple i.v. administrations of high titer retroviral supernatant encoding the HSVtk gene, but not after administration of liposome-complexed plasmid DNA. Using polymerase chain reaction analysis, integration of the provirus was observed in metastasis-bearing lungs (4 of 6 mice) and in the spleens of some ganciclovir-treated animals (2 of 6 mice) but not in the testes, brain, heart, liver, or kidney. The reduction in the number of experimental metastases in C57BL mice exceeded the anticipated extent of transduction of tumor cells, which is indicative of a marked bystander effect. This magnitude of reduction was not observed in immunodeficient athymic mice, suggesting that the immune system plays some part in the bystander effect. In support of these data, we show that, whereas the parental tumor cells are only poorly immunogenic, an effective antitumor immune response is generated following the killing of neoplastic cells in vivo as a result of treatment with ganciclovir. These effects may be responsible for augmenting the efficacy of retroviral infection. The combination of local cell killing by the HSVtk/ganciclovir system and the induction of antitumor immunity suggests new opportunities for the design of vectors for the gene therapy of cancer.

Animals↗
PubMed1994

Targeting of cytokine gene expression to malignant melanoma cells using tissue specific promoter sequences.

BACKGROUND: Transduction of tumor cells in vitro with cDNAs encoding various cytokines and/or immune accessory molecules has been shown to diminish or eliminate tumorigenicity when such cells are returned in vivo to syngeneic animals. One method being explored for in situ gene therapy is to use tissue-specific promoters to direct expression of the therapeutic genes to the tumour cells. DESIGN: This study used the 5' flanking region of the murine tyrosinase gene to direct expression of three different cytokine genes [murine interleukin 2 (IL-2), IL-4 and macrophage colony-stimulating factor (M-CSF)] specifically to murine melanoma cells. RESULTS: Expression of the IL-2 gene, from 2.5 kbp of the 5' flanking region of the murine tyrosinase gene, was detected in 11 out of 55 puromycin-resistant B16 clones isolated after transfection. The highest producing clone secreted 2000 pg/ml/10(6) cells/48 hours as determined by enzyme-linked immunosorbent assay. The IL-2 was tested for biological activity by its ability to stimulate proliferation of the IL-2 dependent CTLL cell line. No detectable level of IL-2 expression occurred in 58 clones of drug-resistant NIH 3T3 cells derived after transfection with the same construct. Similar results were obtained following transfection of these two cell lines with the tyrosinase-IL-4 minigene construct. Expression of IL-2 in the murine melanoma cells completely abrogated their tumorigenicity in syngeneic mice. However, progressively growing tumours were produced from clones in which the IL-2 gene was no longer expressed (as determined by reverse transcriptase polymerase chain reaction). Direct injection of DNA encoding cytokine genes, expressed from the tyrosinase promoter, into established B16 melanomas in syngeneic mice resulted in gene expression within the tumour mass. While no change in tumour growth was observed following such treatment, the results demonstrate that direct injection of naked DNA into a neoplasm can result in uptake and expression of cytokine genes up to 16 days post-injection. CONCLUSION: The use of tissue-specific promoters can limit expression to the required target cell, while the choice of appropriate gene should result in an alteration in tumour burden.

Animals↗
PubMed1994

Targeted therapy for malignant melanoma.

Malignant melanoma is a tumor that offers unique possibilities for approaches to targeted therapy by virtue of the pigment biosynthesis pathway. Such approaches may seek to use the incorporation of toxic intermediates or to use the natural transcriptional control of genes coding for enzymes involved in melanin formation to regulate gene therapy. In this paper, we describe how the 5'-flanking sequences of the genes for tyrosinase or tyrosinase-related protein 1 can be used to drive expression of complementary DNA, coding for immunity-stimulating proteins or for drug-activating enzymes, specifically in melanoma cells. The combination of the tissue specificity resulting from these techniques, coupled with innovative delivery systems, should provide the maximum available therapeutic index and lead to the design of new treatments with limited side effects.

Gene Transfer Techniques↗
PubMed1994

Tumor-specific gene expression.

For gene therapy to become a realistic treatment modality for cancer, it must be at least as effective and, importantly, less toxic than current regimes of radio- and chemotherapy. Therefore, the therapeutic gene(s) must only be delivered to, or expressed in, the appropriate target cells. Whilst delivery vehicles with tropisms restricted at the level of the tumor cell surface are still being developed, protocols have been devised which target gene expression specifically to tumor cells, whilst minimizing expression in surrounding cells. Such tumor-cell specific gene expression can be achieved by any combination of localized gene delivery, the use of vectors with selectivity for dividing cells and incorporation of specific transcriptional regulatory sequences to restrict gene expression predominantly to tumor cells.

Gene Expression Regulation, Neoplastic↗